Proc. Natl. Acad. Sci. USAVol. 80, pp. 5449-5453, September 1983Neurobiology

Geometry and orientation of neuronal processes in cat primaryauditory cortex (A I) related to characteristic-frequency maps

(horseradish peroxidase/tangential organization/tonotopic axes)

RICHARD A. REALE, JOHN F. BRUGGE, AND JIA ZHEN FENGDepartment of Neurophysiology and Waisman Center on Mental Retardation and Human Development, University of Wisconsin, Madison, Wisconsin 53706

Communicated by Clinton N. Woolsey, May 27, 1983

ABSTRACT Microelectrode mapping and horseradish per-oxidase histochemistry were combined to study the relationshipbetween the characteristic-frequency representation and the in-trinsic connectivity of the primary auditory cortex in the cat. Smallextracellular iontophoretic injections of horseradish peroxidasewithin the characteristic-frequency map resulted in labeling ofneuronal processes that, in the tangential plane, radiated outasymmetrically from the injection site over distances of severalmillimeters. The heaviest concentration of labeled fibers was alongan axis parallel with the orientation of the isofrequency line withinwhich the injection had been made. Thus, primary field neuronsthat have the same or a similar characteristic frequency have thepotential of being preferentially interconnected.

The primary auditory cortical field (A I) in the cat contains acomplete and orderly representation of the audible frequencyspectrum (1, 2). Regardless of cortical depth, characteristic fre-quency (CF; i.e., that frequency to which a neuron is most sen-sitive) is relatively constant for neurons recorded in electrodepenetrations normal or near normal to the cortical surface (1,3-5). This representation of tonal frequency changes in an or-derly fashion, from high frequency to low, along the anterior-posterior dimension of A I but is relatively constant along a lineorthogonal to the low-to-high frequency axis. Studies on a va-riety of other species have shown the general applicability ofthis organizational scheme (6-10). In addition to the tonotopicmap, there exists in A I a binaural representation; neurons hav-ing similar binaural properties are also arrayed in vertical col-umns that, when projected to the brain surface, form strips ofcortex oriented oblique or orthogonal to the isofrequency di-mension (11, 12).

In this paper, we address the question of neuronal circuitrywithin A I that may link together elements of these functionalmaps. Specifically, we are interested in the system of intra-cortical fibers that run mainly within, and tangential to, the cor-tical laminae. Studies of Golgi-stained neurons within the mid-dle ectosylvian auditory area have shown that axonal and dendriticfields have preferred tangential orientations (13, 14). The ori-entation patterns observed may bear a relationship to the func-tional maps but, because no electrophysiological mapping hasbeen done in conjunction with the anatomical work, the issueremains unresolved. Here we describe the tangential patternof axonal and dendritic fields of A I neurons, revealed by theuptake of horseradish peroxidase injected extracellularly in verysmall quantities, and show that, indeed, a spatial relationshipexists between these tangential distribution patterns and thetonotopic map determined by microelectrode recording in thesame experiment. The projection patterns may reflect a neu-

ronal circuitry connecting functionally similar, but spatiallyseparate, elements within A I.

MATERIALS AND METHODSObservations were made on six cerebral hemispheres of healthyadult cats anesthetized with sodium pentobarbital (40 mg/kg).The pinnae were removed in such a way that a hollow earpiececould be inserted snugly into each external ear canal and po-sitioned close to the tympanic membrane. These hollow ear-pieces were conduits for sounds generated in our closed acous-tic system, the details of which were described in previous papers(2, 15). The ectosylvian cortical region was exposed and a Lucitechamber was cemented to the skull over the exposed area. Thedura was reflected and a photograph was taken of the exposedbrain, which later served as a guide for visual placement of theelectrode. Glass-insulated tungsten microelectrodes were ad-vanced into the cortex by a Davies microdrive mounted on thechamber, which was filled with mineral oil and hydraulicallysealed to reduce brain pulsations. Recordings were made in asound shielded chamber. Single-unit activity was displayed onan oscilloscope and audibly presented by a loudspeaker. At eachrecording location, the CF was determined for single neuronsor neuron clusters located within middle cortical layers. Binau-ral response properties of neurons were not systematically as-sessed. The site of each penetration was recorded on the brainphotograph. Electrolytic marking lesions (4 ,AA/4 see) wereproduced at selected recording sites to serve as reference pointsin later alignment of the tonotopic map with the map of labeledneurons and neuronal processes. The CF map plotted on thebrain photograph during the experiment was used to guide theplacement of the horseradish peroxidase (HRP) injection. TheHRP [Sigma, type VI, 20% (wt/vol) containing 1% lysophos-phatidylcholine in 0.1 M Tris-HCl buffer] was injected ionto-phoretically (10-20 AA for 15-20 min) through a glass pipette(tip diameter, 25-50 gm).

Twenty-four hours after the HRP injection, the animal wasgiven an overdose of sodium pentobarbital and perfused trans-cardially according to the protocol (procedure II) of Rosene andMesulam (16). After fixation, a slab of tissue containing themapped ectosylvian area and a generous amount of surround-ing cortex, was mounted on the microtome stage. A glass slidewas gently pressed against the slab to flatten the surface of A Iwhile it was freezing. Tissue sections, 50 or 90 ,Am thick, werecut parallel to the flattened surface and processed for HRP his-tochemistry using the cobalt chloride modification (17) of thediaminobenzidine protocol (18, 19), which produces Golgi-likestaining of axons, dendrites, and neuronal perikarya.

Tissue sections, mounted but unstained, were studied under

Abbreviations: A I, primary auditory cortical field; CF, characteristicfrequency; HRP, horseradish peroxidase.

5449

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Proc. Natl. Acad. Sci. USA 80 (1983)

bright- and dark-field illumination. All HRP-labeled neuronalelements visible within several millimeters of the injection sitewere traced under bright-field illumination (magnification, X300)with the aid of a camera lucida. Cortical depth was calculatedfrom microtome settings. The length and distribution of thelabeled processes were determined with the aid of a digitizingtablet interfaced to a Harris 6024/5 computer.

RESULTSCharacteristic frequencies of A I neurons are nearly identicalalong an electrode penetration passing parallel to radially ori-ented cell columns. By projecting the recorded CF to the brainsurface, it is possible to map a two-dimensional CF represen-tation on the flattened ectosylvian cortex. On this surface map,a line joining cortical loci of similar CF values defines an isofre-quency contour. Fig. 1 shows, on such a map, the orientationof three typical isofrequency contours, representing CFs around10, 15, and 21 kHz. In this experiment, a HRP-filled pipetteentered the cortex near the middle of the 15 kHz isofrequencyline. The tip of the pipette was 1,300 gm beneath the pial sur-face during iontophoresis.

Within a radius of 100 ,m, the injection zone is filled withenzyme that has reacted and appears black under-bright-fieldillumination. This blackened core is biconical in shape, ex-tending about 180 gm in either direction, toward the pial sur-face above and the white matter below. Surrounding this core,the HRP reaction product is distinguishable in both neuronal

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reconstructed and displayed on a tangential surface of the right hemi-sphere from brain 8154M. Light interrupted lines in the inset braindrawing show the area in which the characteristic-frequency map wasreconstructed. Characteristic frequencies are expressed in kHz. Largedecimal points are located at recording sites that were projected to thecortical surface. Heavy interrupted lines crossing the cortical surfaceindicate isofrequency contours. Asterisks indicate the physiologicallydetermined boundary between A l and the second auditory area, A II.BR, response over a broad frequency range at lowest threshold inten-sity; o, point at which the HRP-filled pipette entered the cortex.

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Proc. Natl. Acad. Sci. USA 80 (1983) 5451

and nonneuronal elements. Most of the neuronal labeling oc-curs in smooth processes that appear to radiate out from thedark core around the injection site, as shown-in Fig. 2. Theseradiating processes could often be followed for distances ex-ceeding 2 mm in the tangential plane, even for small processesof uniform caliber that often approached the resolution of thelight microscope. Spiny dendrites also contribute heavily to thelabeling pattern but only over a radial distance of some 300 ,mfrom the center of the injection site. The locations of the cellsomata from which specific processes arise are, in general, un-known. Several dozens of labeled neuronal perikarya can oftenbe seen at the injection site. Labeled cells, probably retro-gradely filled with HRP, are also seen in A I several hundredmicrometers away from the dark core of the injection zone. Theirdistribution will be described in a later paper.

Labeled processes were unevenly distributed around the in-jection site. The preferred orientation of labeled processes inthe tangential plane was related to the orientation of frequencyrepresentation on the tonotopic map, regardless of the -depthat which the HRP was injected. Camera lucida tracings fromtwo sections, including the one shown in Fig. 2, are alignedwith the CF map for this animal in Fig. 3. Clearly, more pro-cesses radiate along the dorso-ventral (isofrequency) dimensionof A I than along the antero-posterior dimension. A few fibersare also seen crossing the border between A I and the secondauditory cortical field below. This preferred orientation was alsoapparent in tracings obtained from other tissue sections thatspanned the injection zone.To quantify this tangential distribution, each camera lucida

reconstruction was partitioned into eight pie-shaped bins cen-tered on the core of the injection site. The total length of la-beled processes in each bin was measured. This total in a givenbin was then expressed as a percentage of the largest bin in thehistogram. Two of these normalized histograms, with bin radii

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of 1,200 Am and 600 Am, were constructed for each of threeserially ordered sections in which the labeled processes weremost dense. In this way, we could evaluate contributions to thepreferred orientation from labeled processes located at severalcortical depths relative to the center of the injection site. Fig.4 presents the CF map superimposed on each of the three polarhistogram pairs. Although there are individual differences amongthe histograms, several trends are apparent. First, with one ex-ception (Fig. 4F), the largest bin in each histogram is centeredon the 15-kHz representation of the CF map, which is very closeto the 14-kHz CF recorded at the injection site. HistogramsAC, and E, which include processes traced as far as 1,200 ,mfrom the center of the injection site, are most notable in thisregard; a'15-kHz isofrequency line virtually bisects their largestbins. Second, the smallest bins are located along axes orientednearly orthogonal to the 15-kHz isofrequency line. Third, thepreferred distribution of processes in the tangential plane isgenerally noticeable at distances of 600 ,m from the center ofthe injection site. Fourth, comparison of histograms with dif-ferent bin radii suggests that this orientation does not changeabruptly but remains rather constant within each tissue section.For example, the tangential distributions of labeled processesshown in histograms A and C are quite closely matched by theirpaired histograms B and D, in which only processes located upto 600 ,um were included. Thus, an orientation parallel to the15-kHz representation is maintained for more than 2 mm in thetangential plane in those sections in which the largest bins aredirected both dorsal and ventral to the injection site. In fouradditional experiments, labeled processes were also distributedboth dorsal and ventral to the injection site, along an isofre-quency contour.

In one other brain, an injection in the dorsal one-third of anisofrequency contour produced an asymmetrical radiation oflabeled processes along the isofrequency dimension. Fig. 5

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Neurobiology: Reale et aL

Proc. Natl. Acad. Sci. USA 80 (1983)

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FIG. 4. Polar histogram pairs from tissue sections 1;1 (A and B), 13 (C and D), and 15 (E and F) of brain 8154M are used to relate the tangentialdirections of labeled neuronal processes to the characteristic-frequency map. The total length of labeling in each bin was tabulated and the largestbin was set equal in height to the radius over which the processes were measured. The remaining bins are proportionally smaller. Bin radii are1,200 Am in A, C, and E and 600 am -in B. D, and F. Tissue sections 11, 13, and 15 are respectively located at 1,080 ttm, 1,260 Am, and 1,440 /Ambeneath.the pial surface. Total distance from pial surface to white matter is 1,980 Atm.

presents the CF map for this experiment superimposed on acamera lucida reconstruction of the neuronal labeling in a tissuesection located at the same depth as the center of the injectionsite. The 10-kHz isofrequency contour extends from the injec-

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tion site dorsally approximately 1.2 mm and ventrally about 2.5mm. The normalized histogram shows that the maximal lengthof labeled processes occurs in a dorsally oriented direction alongthe 10-kHz contour. Bin height, representing density of pro-cesses along the ventral extent of the 10-kHz isofrequency line,is no greater, and in some cases is less, than that seen in moreobliquely oriented bins. This pattern was repeated in tissuesections throughout the injection zone.

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DISCUSSION

Through the combined use of anatomical and electrophysio-logical methods, the preferred orientation of neuronal pro-cesses within A I has been directly related to the functional or-

ganization of this field. The greatest density of neuronal processesrelated to a locus in A I is located along the isofrequency di-mension. If these processes make synaptic contact with cellsalong this route, and we have no direct evidence that they do,then neurons or neuronal columns that have the same or a sim-ilar CF, representing the same region of the basilar membrane,are preferentially interconnected. Consequently, with the paucityof fibers running orthogonal to isofrequency lines, neurons withvery dissimilar CFs remain relatively isolated functionally fromone another. Thus, this system of fibers maintains, along withthe thalamocortical (20) and corticocortical (21-23) projectionsystems, a highly segregated cochleotopically organized patternof cortical interconnections.

Glaser et al. (14) showed earlier in Golgi preparations that thedendrites of stellate and pyramidal cells of layer IV and V ofcat auditory cortex, when analyzed by population statistics, havea predominantly dorsal-to-ventral orientation. In our study, wecould not consistently discern a preferred orientation withinthe population of spiny dendritic processes that radiate aboutthe HRP injection site. Rather, most of the labeled processescontributing to the preferred direction more closely resembledthe description of tangentially directed processes seen in fiber-stained preparations of auditory and visual cortices (13, 24, 25).

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Proc. Nati. Acad. Sci. USA 80 (1983) 5453

These tangentially directed fibers run a remarkably- straightcourse and, although their contours are wavy, they do not seemto weave and turn upon themselves. Small lesions of motor cor-tex (26) and visual cortex (25, 27) in monkey have produced ax-onal degeneration patterns that have preferred orientation inthe tangential plane. Similarly, electrophysiologic studies inmotor (28) and somatosensory (29) cortices have shown a pre-ferred tangential orientation of stimulated neuronal elements,suggesting that functionally similar but spatially separate ele-ments within the cortical map are interconnected. More recentintracellular (30) and extracellular (31) HRP studies have shownin greater detail the intrinsic connections of visual cortex. Ax-onal fields there are asymmetric, extending for greater dis-tances along one cortical axis than along another orthogonal toit. Furthermore, these axons do not give off terminal collateralsalong their entire length but instead give them off in clustersso that the intrinsic connections, while widespread, are orga-nized in periodic stripe-like patterns. Mitchison and Crick (32)have proposed that these stripes in visual cortex are related tointrinsic connections among cells with the same orientationpreference. They deduce that the connections might serve togenerate complex or elongated receptive fields that have in-hibitory side bands or end stops depending on the particulartangential direction along which connections are made.

In primary auditory cortex, one such system of "stripes" rep-resents place along the basilar membrane. Along this isofre-quency dimension, neurons having different binaural proper-ties possess a periodic distribution in which irregularly shapedpatches of cortex contain, alternately, aggregations of neuronsexhibiting only one binaural class or another (11, 12, 21, 23).As a consequence, all or most binaural classes of neurons areactivated by any given frequency of stimulation. Whether andto what extent these binaural classes are functionally intercon-nected remains to be determined. However, our observationsof the way in which intracortical fibers ofA I are organized havea seemingly direct consequence-namely, if neurons sharingsimilar binaural properties are preferentially interconnected,then, these interconnections are most widespread along the di-rection in which neurons have the same CF.We are grateful to Mrs. E. Langer for skillful histological assistance,

Ms. S. Hunsaker for excellent photography, and Ms. C. Dizack for carefulillustrations. The work was supported by National Science FoundationGrant BNS76-19893, National Institutes of Health Core Support GrantHD03352, and National Institutes of Health Program Project GrantNS12732.

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